Heat Induces Oxidative Stress in the Testis

In vitro and in vivo experiments suggest that heat stress usually produces ROS, such as superoxide radicals, hydroxyl radicals, and hydrogen peroxide. The generation of ROS occurs constantly, even under physiological conditions, in all living cells, and the rate of free radical generation in the testis or spermatozoa appears to be temperature-dependent [9], For example, the level of spontaneous lipid peroxidation by cultured mouse spermatozoa, as measured by the generation of malondialdehyde (MDA), increases with temperature elevation [10]. In addition, activities of several scavenging enzymes in the testes of rats with experimentally induced cryptorchid-ism were impaired and were accompanied by increased peroxidation of cellular lip-ids [11-13], indicating that testicular oxidative stress is determined by the balance between the generation of ROS and their scavenging systems. Effects of heat stress on cellular components are listed as follows: Cell membrane—changes in fluidity/ stability, alteration in structure, impairment of ion transport, and modulation of the transmembrane efflux pump; cytoplasm—impairment of protein synthesis, denatur-ation of protein structure and function, aggregation of proteins, and induction of heat-shock protein (HSP) synthesis; mitochondria—depolarization of mitochondrial membrane potential, depletion of ATP production, production of ROS, and disruption of Ca2+ transport across the mitochondrial membrane; endoplasmic reticulum (ER)—ER stress from excessive accumulation of misfolded proteins; nucleus— impairment of DNA synthesis, inhibition of DNA repair enzymes, alteration of DNA conformation, and changes in gene expression and signal transduction. In particular, plasma membranes are known to be extremely sensitive to heat stress because of their complex molecular composition of lipids and proteins. Upon temperature elevation, the physical state of lipids changes from a tightly packed gel to a less tightly packed crystalline structure, and the permeability of the cell membrane increases, followed by alteration of the cellular content of several ions (Na+, Mg2+, K+, and Ca2+). For example, influxes of extracellular Ca2+ stimulate the activity of calmodu-lin-dependent protein kinases and inositol triphosphate production [14], resulting in the alteration of intracellular signal transduction cascades. These changes are universal effects observed in all mammalian cells and likely occur in the germ cells. Among the alterations described above, the plasma membrane and mitochondria are considered the major sites of ROS production. Neutrophils mainly produce ROS, leading to deterioration of spermatogenesis in testicular torsion [8, 15]; however, the involvement of neutrophils in the production of heat-induced oxidative stress is unknown. The plasma membrane of testicular cells is rich in polyunsaturated fatty acids and is therefore vulnerable to oxidation by H,O2 and other ROS [12]. The generation of ROS will be augmented in response to elevated metabolism accompanied by oxygen consumption. The content of the redox enzymes, glutathione reductase (GSR) and aldo-keto reductase, which function in the reduction and oxidation of ROS and the resulting carbonyl compounds, is much lower in the germ line cells than in Sertoli and Leydig cells [16]. The antioxidant mechanism against heat-induced oxidative stress is described in the following section. Direct measurements of these gaseous molecules are very difficult to obtain, especially in human testis, and direct and indirect methods to detect seminal ROS are described in other chapters. Stable products of molecules (e.g., nitrite as stable product of nitric oxide (NO) and MDA as a stable product of lipid peroxidation) and modified DNA and proteins (e.g., 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a marker of the oxidation of guanine residues and 4-hydroxy-2-nonenal (4-HNE)-modified proteins as a marker of protein modification by lipid peroxidation products) have been used as surrogate markers of testicular oxidative stress in vivo.

Nitric oxide is a gaseous molecule that is difficult to examine in vivo but is widely examined, because nitric oxide synthase (NOS) expression is easy to investigate at the RNA and protein levels. At higher concentrations, NO promotes germ cell apoptosis through production of peroxynitrite by a reaction between NO and superoxide [17]. The tissue concentration of NO is determined by the type of NOS involved: inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Basically, tissue concentrations of NO produced by iNOS are 10- to 100fold higher than those from eNOS and nNOS and can cause necrosis of germ cells [15]. Increased NO synthesis through up-regulation of iNOS by heat stress has been implicated in cellular injury and apoptosis in various cell systems. Heat treatment (43°C for 30 min for 2 consecutive days) markedly induced iNOS expression in testicular germ cells of Cynomolgus monkeys after 3-8 days [18]. Increased iNOS immunoreactivity was noted in heat-susceptible germ cells (pachytene spermato-cytes and round spermatids) 3 days after treatment. On day 28, iNOS expression in germ cells is similar to that of controls, whereas very high iNOS expression was noted in Sertoli cells, because the majority of apoptotic germ cells within this time frame had been lost through phagocytosis [19, 20]. eNOS was observed mainly in Sertoli cells and spermatogonia in rats. No obvious alteration in eNOS levels was detected in any of the heat-treatment groups [18]. In humans, NO, which is basically a vasodilator, is shown to be produced by eNOS in endothelial cells in patients with varicocele [21].In eNOS transgenic mice, NO produced through eNOS is reported to play a functional role in spermatogenesis in cryptorchid-induced apoptosis [22] , whereas a several-fold increase in the levels of NO produced by eNOS is less likely to cause oxidative stress.

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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